In the two years since the installation of the Vaccine Branch Astrios-EQ (installed mid-2014, in conjunction with a CRADA that we initiated with Beckman-Coulter in 2013), we developed nanoFACS protocols for fluorescence detection and calibration, for counting, and for high-throughput sorting, to sort EV subsets from plasma and other biofluids by nanoFACS. Using multiple distinct vesicle populations, we find that nanoFACS sorting is uniquely able to produce high fidelity EV subsets (95-97% pure), with functional biological cargo (RNA and protein). During the past 9-10 months, our focus has been exclusively on application of this method for clinically important studies, for the study of cancer disease burden, metastatic potential, and responses to treatment. Our NanoFACS approach that we developed as part of this ACI program is the only method currently able to analyze and preparatively sort functional EVs based on single EV characteristics, rather than bulk attributes. Furthermore, we have devised a new class of labels (Molecular NanoTags) that can be used with nanoFACS, not only to improve the detection of EV epitopes, but also to enable single molecule counting by flow cytometry. The specialized instrumentation and expertise assembled for this nanoFACS program does not exist anywhere else in the world, is a distinctive asset for NCI. We now have a unique opportunity to leverage this nanoFACS program in the Clinical Center to demonstrate the potential impact of this approach for oncology, immunology, and personalized medicine. Toward the goal of utilizing nanoFACS in the study of tumor-derived EVs as a liquid biopsy, we have tested and confirmed nanoFACS resolution of cancer and cancer stem-cell markers: PSMA, CD147, CD44, and CD26. During the past 6 months, we also found that immune cell markers such as MHC-II and PD-L1 identify distinct EV subsets. We predict that the co-receptors and miRNA that are expressed on or contained as cargo of MHC-II EV subsets will correlate with immune responses- of the cells or the organism that produced those EVs. We have validated PD-L1 detection on cell line-derived EVs and are beginning to study detection of PD-L1 on biofluid-derived EVs. We are working with James Gulley and Tim Greten to determine whether circulating PD-L1 levels on tumor-derived EVs or antigen presenting cell-derived EVs correlate with tumor biopsy levels of PD-L1 and responses to PD-L1/PD-1-directed therapies. Similarly, we are working now with members of the Senior Prostate Group to determine whether tumor-derived EVs with PSMA, EpCAM, and CD147 correlate with prostate cancer disease burden, prognosis, and responses to treatment.